Proteases/Subtilisins
Proteases, or (interchangeably) peptidases, are enzymes that cleave the amide linkages in protein substrates. Bacteria of the Bacillus species secrete two extracellular species of protease, a neutral or metalloprotease, and an alkaline protease which is functionally a serine endopeptidase, referred to as subtilisin.
A serine protease is an enzyme which catalyses the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site White, Handler and Smith (1973); Principles of Biochemistry; Fifth Edition, McGraw-Hill Book Company, N.Y., 271-272!.
The bacterial serine proteases have molecular weights in the range of 20,000 to 45,000. They hydrolyse simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 for review, see Priest; Bacteriological Rev., 41 711-753 (1977)!.
In relation to the present invention a subtilisin is a serine protease produced by Gram-positive bacteria or fungi. According to another definition, a subtilisin is a serine protease, wherein the relative order of the amino acid residues in the catalytic triad is Asp--His--Ser (positions 32, 64, and 221). A wide variety of subtilisins has been identified, and the amino acid sequences of a number of subtilisins have been determined. These include among others six subtilisins from Bacillus strains, namely, subtilisin 168, subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, and mesentericopeptidase Kurihara et al. (1972); J. Biol. Chem.; 247 5629-5631; Wells et al. (1983); Nucleic Acids Res.; 11 7911-7925; Stahl and Ferrari (1984); J. Bacteriol.; 159 811-819; Jacobs et al. (1985); Nucl. Acids Res.; 13 8913-8926; Nedkov et al. (1985); Biol. Chem. Hoppe-Seyler; 366 421-430; Svendsen et al. (1985); FEBS LETTERS; 196 228-232! one subtilisin from an actino-mycetales, Thermitase from Thermoactinomyces vulgaris Meloun et al. (1985); FEBS LETTERS; 1983 195-200!, and one fungal subtilisin, proteinase K from Tritirachium album Jany and Mayer (1985); Biol. Chem. Hoppe-Seyler; 366 584-492!.
Proteases such as subtilisins have found much utility in industry, particularly in detergent formulations, as they are useful for removing proteinaceous stains.
The Structure of Proteins
Proteases are globular proteins and quite compact due to the considerable amount of folding of the long polypeptide chain. The polypeptide chain essentially consists of the "backbone" and its "side-groups". As the peptide bond is planar, only rotations around the C.sub.a --N axis and the C.sub.a --C' axis are permitted. Rotation around the C.sub.a --N bond of the peptide backbone is denoted by the torsion angle .phi. (phi), rotation around the C.sub.a --C' bond by .psi. (psi) vide e.g. Creighton, T. E. (1984); Proteins; W. H. Freeman and Company, New York!. The choice of the values of these angles of rotation is made by assigning the maximum value of +180.degree. (which is identical to -180.degree.) to the maximally extended chain. In the fully extended polypeptide chain, the N, C.sub.a and C' atoms are all "trans" to each other. In the "cis" configuration, the angles .phi. and .psi. are assigned the value of 0.degree.. Rotation from this position around the bonds so that the atoms viewed behind the rotated bond move "counter-clockwise" is assigned negative values by definition, those "clockwise" are assigned positive values. Thus, the values of the torsion angles lie within the range -180.degree. to +180.degree..
Since the C.sub.a -atoms are the swivel point for the chain, the side-groups (R-groups) associated with the C.sub.a -atoms become extremely important with respect to the conformation of the molecule.
The term "conformation" defines the participation of the secondary and tertiary structures of the polypeptide chains in moulding the overall structure of a protein. The correct conformation of a protein is of prime importance to the specific structure of a protein and contributes greatly to the unique catalytic properties (i.e. activity and specificity) of enzymes and their stability.
The amino acids of polypeptides can be divided into four general groups: nonpolar, uncharged polar, and negatively or positively charged polar amino acids. A protein molecule, when submerged in its aqueous environment in which it normally occurs, tends to expose a maximum number of its polar side-groups to the surrounding environment, while a majority of its nonpolar side groups is oriented internally. Orientation of the side-groups in this manner leads to a stabilization of protein conformation.
Proteins, thus, exist in a dynamic equilibrium between a folded and ordered state, and an unfolded and disordered state. This equilibrium in part reflects the short range interactions among the different segments of the polypeptide chain, which tends to stabilize the overall structure of proteins. Thermodynamic forces simultaneously tend to promote randomization of the unfolding molecule.
A way to engineer stabilized proteins is to reduce the extent of unfolding by decreasing the flexibility of the polypeptide backbone, and simultaneously decreasing the entropy of the unfolded chain. So far only few attempts have been made to implement this rationale in the development of novel stabilized proteases.
A general principle of increasing protein thermostability has been provided Suzuki, Y. (1989); Proc. Japan Acad.; 65 Ser. B!. In this article Suzuki states that the thermostability of a globular protein can be enhanced cumulatively to a great extent by increasing the frequency of proline occurrence at the second site of .beta.-turns without significant alterations in the secondary and tertiary structures as well as in the catalytic function of enzymes. The principle is based on various facts and findings, among these the fact that proline residues show a strong tendency to occur preferentially at the second site of .beta.-turns Levitt, M (1978); Biochemistry; 17 4277-4285; and Chou, P. Y. & Fasman, G. D. (1977); J. Mol. Biol.; 115 135-175!. The principle is restricted to insertion of proline into the second site of .beta.-turns in proteins, no other sites are mentioned.
International Patent Publication WO 89/01520 (Cetus Corporation, USA) provides a method for increasing the stability of a protein by decreasing the configurational entropy of unfolding the protein. The method is applied on a Streptomyces rubiqinosus xylose isomerase, and it involves substitution of an amino acid with proline, or replacement of glycine with alanine, at predicted substitution sites.
In International Patent Publication WO 89/09819 (Genex Corporation, USA) a method for combining mutations for stabilization of subtilisins is provided. This publication lists a number of amino acid mutations that have been found to be thermally stabilizing mutations. The list comprises substitution of serine with proline at position 188 of subtilisins (BPN' numbering).
International Patent Publication WO 87/05050 (Genex Corporation, USA) describes a method for mutagenesis and screening. By this method one or more mutations are introduced by treatment with mutagenizing agents, and the method includes subsequent screening for products with altered properties. As a result of this random mutagenesis a subtilisin with a proline residue at position 188 (BPN' numbering) is provided.
It is an object of this invention to provide novel proteases having improved stability.